The basic structural unit of oligonucleotide is nucleotide which comprises base, pentose and phosphoric acid. Nucleotides may be classified into RNA (ribonucleic acid) and DNA (deoxyribonucleic acid) which have the same basic chemical structure, except that they contain different pentose in which the pentose of RNA is D-ribose, and the pentose of DNA is D-2-deoxyribose. The base generally includes guanine, adenine, cytosine, thymine and uracil. The chemical synthesis method of oligonucleotides currently widely adopted is solid phase synthesis method (also known as phosphoramidite-triester method). At pages 520-521 of the Biochemistry (Book 1, Edition 3, Wang Jingyan et al, Higher Education Press), the principle of solid phase synthesis is introduced as follows: All reactions take place in a solid phase column which has a small synthesis scale. Firstly, some free groups on the nucleotides to be activated are protected (blocked) to make the reactions go towards the designed direction. 5′-OH is protected with DMTr (4,4′-dimethoxytriphenylmethyl) group, and the amino groups on the base are protected with benzoyl groups. 3′-OH is activated with amino phosphorous acid compound. The 3′-OH of the first nucleotide is combined with the solid phase (resin). A phosphite triester is formed between its 5′-OH and the activated monomer (nucleotide). As the 5′-OH on the activated monomer and the amino group on the base are protected, they won't participate in the reaction. During the reaction in the second step, phosphite triester is oxidized by iodine into phosphate triester; during the reaction in the third step, trichloroacetic acid is added to remove protective agent DMTr on 5′-OH in the growing chain. By now, DNA chain has been extended by one more nucleotide unit and is ready for the reactions for next round of extension. According to the program input in advance, after the synthesis of a whole DNA fragment is completed, thiophenol is applied to remove the protective agent DMTr on 5′-OH, and concentrated ammonium hydroxide is applied to disconnect DNA fragment from the solid phase resin so that DNA is eluted. Then concentrated ammonium hydroxide is applied again to remove the protective agent on the base under heated condition. Finally ammonium hydroxide is removed under vacuum.
As the whole process of the foregoing solid phase synthesis is conducted in a solid phase column, due to the limitation of the volume of the solid phase column, this method has a small synthesis scale and low yield and can't meet the requirement of mass synthesis. Following the deepening of the research on nucleic acid, on the one hand, the amount of the oligomeric RNA applied in scientific research increases rapidly, and on the other hand, the clinical application of RNA is in the ascendant, so it is a matter of significance to design a method for synthesizing RNA on a large scale.